Nuclear FGFR1 regulates gene transcription and promotes antiestrogen resistance in ER+ breast cancer

FGFR1 overexpression has been associated with endocrine resistance in ER+ breast cancer. We found FGFR1 localized in the nucleus of breast cancer cells in primary tumors resistant to estrogen suppression. We investigated a role of nuclear FGFR1 on gene transcription and antiestrogen resistance. Tumors from patients treated with letrozole were subjected to Ki67 and FGFR1 IHC. MCF7 cells were transduced with FGFR1(SP-)(NLS) to promote nuclear FGFR1 overexpression. FGFR1 genomic activity in ER+/FGFR1-amplified breast cancer cells ± FOXA1 siRNA or ± the FGFR tyrosine kinase inhibitor (TKI) erdafitinib was examined by ChIP-Seq and RNA-Seq. The nuclear and chromatin-bound FGFR1 interactome was investigated by Mass Spectrometry (MS). High nuclear FGFR1 expression in ER+ primary tumors positively correlated with post-letrozole Ki67 values. Nuclear FGFR1 overexpression influenced gene transcription and promoted resistance to estrogen suppression and to fulvestrant in vivo. A gene expression signature induced by nuclear FGFR1 correlated with shorter survival in the METABRIC cohort of patients treated with antiestrogens. ChIP-Seq revealed FGFR1 occupancy at transcription start sites, overlapping with active transcription histone marks. MS analysis of the nuclear FGFR1 interactome identified phosphorylated RNA-Polymerase II and FOXA1, with FOXA1 RNAi impairing FGFR1 recruitment to chromatin. Treatment with erdafitinib did not impair nuclear FGFR1 translocation and genomic activity. These data suggest nuclear FGFR1 contributes to endocrine resistance by modulating gene transcription in ER+ breast cancer. Nuclear FGFR1 activity was unaffected by FGFR TKIs, thus supporting the development of treatment strategies to inhibit nuclear FGFR1 in ER+/FGFR1 overexpressing breast cancer

Role of Spo13 in regulating meiotic chromosome segregation in yeast

During the mitotic cell cycle, DNA replication and chromosome segregation strictly alternate in order to maintain a constant ploidy. Meiosis, on the other hand, is a special form of cell division that is characterized by a single round of DNA replication followed by two consecutive nuclear divisions. In order to understand how these two divisions are implemented, mutants that undergo a single meiotic division are of special interest. The spo13 mutant undergoes a single nuclear division, during which it segregates a mixture of homologous chromosomes and sister chromatids. The Spo13 protein has been implicated in both the monopolar attachment of sister chromatids at meiosis I and the protection of centromeric cohesin until the second meiotic division. However, we showed that SPO13 deletion cells were delayed in metaphase I by the spindle assembly checkpoint (SAC). In this work, we re-investigated the involvement of Spo13 for monopolar attachment and centromeric cohesin protection while taking into account the delay at metaphase I. We found that, while Spo13 is directly required for monopolar attachment, it is not directly involved in centromeric cohesin protection. Indeed, the premature loss of centromeric cohesin in spo13 cells is due to their delay in metaphase I, and shortening this delay restores centromeric cohesin protection. Furthermore, we found that Spo13, together with the polo-like kinase, prevents the activation of APC/CAma1 in meiosis I by phosphorylating the B-type cyclin, Clb1. Spo13 is therefore required for coordinating the APC/C activity with meiosis I-specific events

The meiosis-specific Cdc20 family-member Ama1 promotes binding of the Ssp2 activator to the Smk1 MAP kinase.

Smk1 is a meiosis-specific MAP kinase (MAPK) in budding yeast that is required for spore formation. It is localized to prospore membranes (PSMs), the structures that engulf haploid cells during meiosis II (MII). Similar to canonically activated MAPKs, Smk1 is controlled by phosphorylation of its activation-loop threonine (T) and tyrosine (Y). However, activation loop phosphorylation occurs via a noncanonical two-step mechanism in which 1) the cyclin-dependent kinase activating kinase Cak1 phosphorylaytes T207 during MI, and 2) Smk1 autophosphorylates Y209 as MII draws to a close. Autophosphorylation of Y209 and catalytic activity for substrates require Ssp2, a meiosis-specific protein that is translationally repressed until anaphase of MII. Ama1 is a meiosis-specific targeting subunit of the anaphase-promoting complex/cyclosome that regulates multiple steps in meiotic development, including exit from MII. Here, we show that Ama1 activates autophosphorylation of Smk1 on Y209 by promoting formation of the Ssp2/Smk1 complex at PSMs. These findings link meiotic exit to Smk1 activation and spore wall assembly

A Novel Mechanism for CTCF in the Epigenetic Regulation of Bax in Breast Cancer Cells

Wepreviously reported the association of elevated levels of themultifunctional transcription factor, CCCTC binding factor (CTCF), in breast cancer cells with the specific anti-apoptotic function of CTCF. To understand the molecularmechanisms of this phenomenon, we investigated regulation of the human Bax gene by CTCF in breast and non-breast cells. Two CTCF binding sites (CTSs) within the Bax promoter were identified. In all cells, breast and non-breast, active histone modifications were present at these CTSs, DNA harboring this region was unmethylated, and levels of Bax mRNA and protein were similar. Nevertheless, up-regulation of Bax mRNA and protein and apoptotic cell deathwere observed only in breast cancer cells depleted of CTCF.We proposed that increased CTCF binding to the Bax promoter in breast cancer cells, by comparison with non-breast cells, may be mechanistically linked to the specific apoptotic phenotype in CTCF-depleted breast cancer cells. In this study, we show that CTCF binding was enriched at the Bax CTSs in breast cancer cells and tumors; in contrast, binding of other transcription factors (SP1,WT1, EGR1, and c-Myc) was generally increased in non- breast cells and normal breast tissues. Our findings suggest a novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells, whereby elevated levels of CTCF support preferential binding of CTCF to the Bax CTSs. In this context, CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis. © 2013 Neoplasia Press, Inc

Dynamical modeling of the network controlling meiotic divisions

Mitosis and meiosis are both controlled by oscillations in the activities of cyclin- dependent kinase 1 (Cdk1) and the anaphase-promoting complex/cyclosome (APC/C). Nevertheless, these types of cell division differ in fundamental aspects. In mitosis, Cdk1 and APC/C-Cdc20 form a cyclical system whereby each cycle recreates the starting conditions for the next one. As a result, chromosomes duplication during S-phase alternates with chromosome segregation during M-phase. By contrast, meiosis is a linear pathway of precisely two waves of Cdk1 and APC/C-Cdc20 activity that govern the progression through one S-phase followed by two M-phases and a differentiation program dedicated to the formation of gametes or spores. Despite recent advances in our understanding of meiosis, it is unclear how the mitotic cell cycle engine is modified to regulate the two meiotic divisions. Therefore, we combined mathematical modeling with experimental studies on budding yeast to describe the general mechanism of progression through meiotic divisions with special emphasis on the regulation of the exit from meiosis II. We showed that progression through meiotic divisions is driven by a well conserved Cdk1-APC/C-Cdc20 oscillator complemented by a set of meiotic regulators in order to perform two, and only two, meiotic divisions. The machinery that terminates the oscillations after completion of meiosis II consists of a meiosis I-specific mechanism that unleashes the irreversible inactivation of M-phase regulators after the second wave of APC/C-Cdc20 activity, thereby preventing cells from undergoing an additional third division. Here, we describe the roles of the two main APC/C co- activators, Ama1 and Cdc20, in triggering the exit from meiosis and in terminating the oscillations. We show that Ama1 acts as a terminator of the meiotic oscillations, while Cdc20 is important for the proper timing of the exit from meiosis II. We propose that in the absence of Ama1, the properties of the system change, allowing Cdc20 to adopt the function of the terminator precisely after meiosis II. In addition, we evaluate an APC/C-independent mechanisms, which might be important for preventing a third meiotic division

Association of FGFR1 with ER-alpha maintains ligand-independent ER transcription and mediates resistance to estrogen deprivation in ER+ breast cancer

In a cohort of patients with ER+ breast cancer, tumors with FGFR1 amplification retained high proliferation upon estrogen deprivation with the aromatase inhibitor letrozole. Estrogen deprivation increased total and nuclear FGFR1 and FGF ligands in ER+/FGFR1-amplified primary tumors and breast cancer cells. In estrogen-free conditions, FGFR1 associated with ER-alpha in tumor cell nuclei and regulated the transcription of ER-dependent genes. This interaction and transcriptional output were inducible by FGF ligands and blocked by a kinase-dead FGFR1 mutant or FGFR kinase inhibitors. ChIP-seq of FGFR1 amplified cells treated with FGF3 showed binding of FGFR1 and ERα to DNA. Treatment with the ER downregulator fulvestrant and/or the FGFR inhibitor lucitanib reduced binding of ERα or FGFR1 to DNA. RNA-seq data from FGFR1-amplified patients’ tumors treated with letrozole showed enrichment of estrogen response and E2F target genes. Finally, growth of ER+/FGFR1-amplified breast cancer cells and patient-derived xenografts was more potently inhibited by fulvestrant and lucitanib combined than each drug alone, suggesting a causal association of aberrant FGFR signaling with endocrine resistance. These data suggest the ER-alpha pathway is still active in estrogen-deprived ER+/FGFR1-amplified breast cancers and, therefore, these tumors should be considered for treatment with a combination of ER and FGFR antagonists

Cholesterol metabolism in breast cancer: Prognostic factors and optimizing treatment

AbstractBreast cancer (BC) is the most common cancer among women in Sweden and worldwide. Adjuvant endocrine therapies are effective, yet 20-30% patients experience disease relapse. Altered cholesterol metabolism is an emerging hallmark of breast cancer proliferation and endocrine therapy resistance. Studies have shown that the use of cholesterol-lowering medications (statins) reduces the risk of disease recurrence in a subset of patients. However, the patterns of breast cancer recurrence remains poorly understood, and treatment predictive biomarkers are needed. 27-hydroxycholesterol (27HC), an oxysterol synthesized from cholesterol by the enzyme CYP27A1, is a selective estrogen receptor (ER) modulator. It has been shown that 27HC is an important mediator that links cholesterol and breast cancer pathology. Aims: The main aims of this thesis were i) to explore the molecular features associated with the heterogeneous response of BC cells to statin treatment alone or in combination with endocrine therapy, ii) investigate the patterns of BC recurrence in patients with post-diagnosis statin use, and iii) characterize CYP27A1 expression in primary BC in relation to tumor pathological features and prognosis. Methods: In papers I and V, BC cell lines were treated with statins and/or endocrine treatments to study its impact on cell growth, lipid metabolism, endocrine signaling, and other metabolic pathways using a combination of global transcriptional profiling and molecular assays. In paper II, the prognostic impact of statin use on disease recurrence was investigated in the Malmö Diet and Cancer Study Cohort. Disease recurrence rates were compared between post-diagnosis statin users versus non users using Cox regression models to compute crude and adjusted hazard ratios. In paper III and IV, the prognostic impact of CYP27A1 expression, as measured by immunohistochemistry and/or RNAscope, was evaluated in three independent cohorts of BC patients. In paper IV, the impact of 27HC on BC cell proliferation was measured under lipid depleted conditions.Results: Statin treatment induced a dose- and time-dependent accumulation of intracellular lipid droplets and significantly upregulated the expression of Stearoyl-CoA desaturase—a key regulator of lipid synthesis in statin-insensitive cell lines. Statin use was associated with a reduced risk of distant BC recurrence, but no association between loco-regional recurrence was found. CYP27A1 is a prognostic biomarker with differential impact based on patients’ menopausal status. High CYP27A1 protein expression was independently associated with aggressive tumor characteristics and predicted for inferior overall survival for postmenopausal patients with ER+ BC. Elevated expression of CYP27A1, both at the protein and transcript levels, was associated with high histological grade and high Ki67 expression and was independently prognostic for superior survival outcomes in premenopausal BC patients with node-negative disease. BC cells have heterogeneous response to statin and endocrine combination therapy. Long-term atorvastatin treatment upregulated ER expression in tamoxifen-resistant MCF7 cells while the expression of cyclin D1, E cadherin, and Snail were downregulated in endocrine therapy-resistant MCF7 cells treated with atorvastatin. The gene expression of CYP27A1 was downregulated upon addition of atorvastatin in long-term estrogen-deprived MCF7 cells.Conclusions: Taken together, our results indicate that statins induce heterogeneous anti-proliferative and transcriptional activity in BC cell lines. Statins reduce distant disease recurrence in a subgroup of BC patients. High CYP27A1 is a poor prognostic marker in post-menopausal BC patients. Long term statin and estrogen deprivation reduced CYP27A1 expression in MCF7 cell line. Further studies to assess the clinical utility of CYP27A1 as a treatment predictive marker for statin use are warranted

Analysis of Paired Primary-Metastatic Hormone-Receptor Positive Breast Tumors (HRPBC) Uncovers Potential Novel Drivers of Hormonal Resistance

We sought to identify genetic variants associated with disease relapse and failure to hormonal treatment in hormone-receptor positive breast cancer (HRPBC). We analyzed a series of HRPBC with distant relapse, by sequencing pairs (n = 11) of tumors (primary and metastases) at >800X. Comparative genomic hybridization was performed as well. Top hits, based on the frequency of alteration and severity of the changes, were tested in the TCGA series. Genes determining the most parsimonious prognostic signature were studied for their functional role in vitro, by performing cell growth assays in hormonal-deprivation conditions, a setting that mimics treatment with aromatase inhibitors. Severe alterations were recurrently found in 18 genes in the pairs. However, only MYC, DNAH5, CSFR1, EPHA7, ARID1B, and KMT2C preserved an independent prognosis impact and/or showed a significantly different incidence of alterations between relapsed and non-relapsed cases in the TCGA series. The signature composed of MYC, KMT2C, and EPHA7 best discriminated the clinical course, (overall survival 90,7 vs. 144,5 months; p = 0.0001). Having an alteration in any of the genes of the signature implied a hazard ratio of death of 3.25 (p<0.0001), and early relapse during the adjuvant hormonal treatment. The presence of the D348N mutation in KMT2C and/or the T666I mutation in the kinase domain of EPHA7 conferred hormonal resistance in vitro. Novel inactivating mutations in KMT2C and EPHA7, which confer hormonal resistance, are linked to adverse clinical course in HRPBC

The ability to generate differentiated and senescent progeny is a major determinant of breast cancer heterogeneity

Ankara : The Department of Molecular Biology and Genetics and the Institute of Engineering and Science of Bilkent University, 2009.Thesis (Ph. D.) -- Bilkent University, 2009.Includes bibliographical references leaves 113-123.Breast cancer displays distinct subtypes, such as luminal A, luminal B, and basallike. The prognosis and therapeutic response of each subtype is different. The mechanisms involved in the generation of these tumor types are poorly understood. Our aim was to test whether the ability to generate senescent progeny contributes to breast cancer heterogeneity. A panel of 12 breast cancer cell lines, 31 isogenic clones, and 12 breast tumors were used. We classified breast cancer cell lines into senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All ER+ cell lines tested and some ER-positive (ER+) breast tumors displayed senescence. Acute loss and tamoxifen-mediated inactivation of ER triggered a robust senescence response in SCP type T47D cell line. In contrast, ER-overexpression, estrogen treatment and p21Cip1 knockdown inhibited senescence. Neutralization of reactive oxygen species also abolished senescence. Breast cancer cell subtypes displayed divergent ability to produce differentiated progeny. The SCP subtype cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some cell lines generated only CD44+/CD24-/ ER-/ASMA- progenitor/stem-like cells, and others only CD24+/ERluminal-like, but not ASMA+ myoepithelial-like cells. SCP cell lines were less tumorigenic, and they clustered with luminal A/normal like tumors. In contrast, ICP subtypes were more tumorigenic, and they clustered together with basal/luminal B tumors. Our results show that breast cancer cell lines clustering with luminal A/normal-like and basal/luminal B tumors respectively, differ from each other by the ability to generate differentiated and senescence-arrested progeny.Mumcuoğlu, MinePh.D